Endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on vascular endothelium. ESG is composed of membrane glycoproteins, glycosaminoglycans and proteoglycans, forming a bulky, matrix-like structure that serves critical functions in mechanotransduction of blood flow, maintenance of the endothelial permeability, and the control of leukocyte adhesion and inflammation. One of the most important normal physiological functions of ESG is to mediate mechanotransduction that leads to the intake of calcium ions and the production of Nitric Oxide (NO) in response to blood flow. Dysfunctional ESG mechanotransduction has been found in cardiovascular diseases such as sepsis, ischemia-reperfusion, hypertension, and diabetes. While the critical involvement of ESG in cardiovascular diseases has been established, its biomechanical properties, as well as the mechanisms underlying its normal mechanotransduction, have resisted elucidation. This lack of progress is due in large part to the fact that mechanical forces and responses that occur at the molecular and sub-cellular levels are transient, minute and therefore difficult to trace and measure. The goal of the current proposal is to develop new research tools and model systems, and use them to uncover the biomechanical and mechanotranduction properties of ESG. We will characterize mechanisms underlying ESG-mediated mechanotransduction on a single-cell level using a novel Atomic Force microscopy (AFM)-fluorescence microscopy approach. The proposed study will achieve a clearer understanding of ESG-mediated mechanotransductory function, with important implications for ESG-related diseases, such as sepsis, ischemiareperfusion, diabetes and hypertension, and their therapeutics.